Genes and their encoded proteins which regulate gene expression of the interleukin-2 receptor and of human lymphotropic retroviruses

ABSTRACT

The present invention is directed to genes, termed Rpt-1 (regulatory protein T lymphocyte-1), which are expressed at higher levels by resting CD4 +  helper/inducer T cells relative to activated CD4 +  cells. The invention also relates to the proteins encoded by such genes, termed rpt-1 proteins, which regulate gene expression directed by the promoter region of the interleukin-2 receptor (IL-2r) alpha chain gene or by the promoter region of the long terminal repeat of human lymphotropic retroviruses such as the human immunodeficiency virus type 1 (HIV-1) human T cell leukemia virus (HTLV)-I, and HTLV-II. In particular, rpt-1 proteins down-regulate gene expression controlled by the promoter of the IL-2r alpha chain gene or by the promoter of the long terminal repeat of HIV-1. The proteins and nucleic acids of the invention have value in diagnosis and therapy of immune disorders such as AIDS. 
     In a specific example of the present invention, an Rpt-1 gene and its encoded intracellular protein of approximately 41,000 daltons molecular weight are described. The rpt-1 protein is shown to be selectively expressed by activated CD4 +  T cells, and to down-regulate gene expression of the IL-2r and the HIV-1.

Pursuant to the provisions of 35 U.S.C. §202(c), it is herebyacknowledged that the Government has certain rights in this invention,which was made in part with funds from the National Institutes ofHealth.

TABLE OF CONTENTS

1. Introduction

2. Background of the Invention

2.1. T Cell Gene Expression

2.2. The Interleukin-2 Receptor

2.3. Human Immunodeficiency Virus Type I and Other Human Retroviruses

3. Summary of the Invention

3.1. Definitions

4. Description of the Figures

5. Detailed Description of the Invention

5.1. Isolation of the Rpt-1 Gene

5.2. Expression of the Rpt-1 Gene

5.3. Identification and Purification of the Expressed Gene Product

5.4. Structure of the Rpt-1 Gene and Protein

5.4.1. Genetic Analysis

5.4.2. Protein Analysis

5.5 Regulation of Gene Expression of the Interleukin-2 Receptor and ofHuman Lymphotropic Retroviruses

5.6. Anti-Rpt-1 Antibody Production

5.7. Rpt-1 Related Derivatives, Analogues, and Peptides

5.8. Uses of Rpt-1

5.8.1. Diagnosis

5.8.2. Therapy

6. Cloning and Characterization of the Rpt-1 Gene and its EncodedProtein

6.1 Materials and Methods

6.1.1. Cells

6.1.2. Activation of T Cells

6.1.3. Production of a T Cell Probe

6.1.4. Construction of a T Cell cDNA Library

6.1.5. Nucleic Acid Blotting and Hybridization

6.1.6. Western Blot Analysis

6.1.7. Plasmids

6.1.8. Transfection of Cell Lines and Chloramphenicol Acetyl TransferaseAssays

6.1.9. Immunofluorescence

6.2. Results

6.3. Discussion

7. Deposit of Microorganisms

1. INTRODUCTION

The present invention is directed to genes, termed Rpt-1 (regulatoryprotein T lymphocyte-1), which are expressed at higher levels by restingCD4⁺ inducer T cells relative to activated CD4⁺ cells. The inventionalso relates to proteins encoded by such genes, termed rpt-1 proteins,which regulate gene expression directed by the promoter region of theinterleukin-2 receptor alpha chain gene or by the promoter region of thelong terminal repeat of human lymphotropic retroviruses. The proteinsand nucleic acids of the invention have value in diagnosis and therapyof immune disorders such as AIDS.

2. BACKGROUND OF THE INVENTION

2.1. T Cell Gene Expression

Analysis of cellular and viral proteins produced by clones of inducer,cytotoxic, and suppressor T cells, has shown that each T cell subset isgenetically programmed to specify particular patterns of proteinsynthesis before and after activation by antigen (Nabel, G., et al.,1981, Cell 23:19-28; Fresno, M., et al., 1982, Cell 30:707-713; Zagury,D., et al., 1986, Science 231:860-863). For instance, the levels ofexpression of the retrovirus associated with the acquiredimmunodeficiency syndrome (AIDS), HIV-1, are markedly increased uponactivation of infected inducer T cells (Zagury, D., et al., 1986,Science 231:860-863; Klatzmann, D. and Gluckman, J.C., 1986, Immunol.Today 7:291-296; Nabel, G. and Baltimore, D., 1987, Nature 326:711-713;Tong-Starksen, S. E., et al., 1987, Proc. Natl. Acad. Sci. U.S.A.84:6845). An intracellular protein that regulates genes expressed inresting and activated T cells has been described (e.g., Nabel, G. andBaltimore, D., 1987, Nature 326:711-713; Tong-Starksen, S. E., et al.,1987, Proc. Natl. Acad. Sci. U.S.A. 84:6845).

2.2. The Interleukin-2 Receptor

T cells secrete a variety of polypeptides affecting immunoregulation ofhematopoietic cells and are themselves subject to regulation by hormonepeptides interacting with specific receptors on their cell surface.Interleukin-2, originally termed T cell growth factor, is synthesizedand secreted by antigen- or lectin-activated T lymphocytes in thepresence of macrophage-derived interleukin-1, and interacts withspecific high-affinity membrane receptors to exert its biologicaleffects (Smith, K. A., 1980, Immunol. Rev. 51:337-357; Leonard, W. J.,et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:6957-6961). Theinterleukin-2 receptor (IL-2r, Tac antigen) is not present on thesurface of resting T or B lymphocytes. Upon activation by specificantigens or mitogens, T cell proliferation is mediated by an autocrinemechanism whereby activated cells secrete interleukin-2 (IL-2) and alsoexpress cell surface receptors for IL-2 (IL-2r) (Mier, J. W. and Gallo,R. C., 1980, Proc. Natl. Acad. Sci. U.S.A. 77:6134; Robb, R. J., et al.,1981, J. Exp. Med. 154:1455; Leonard, W. J., et al., 1982, Nature300:267; Meuer, S. C., et al., 1984, Proc. Natl. Acad. Sci. U.S.A.81:1509; Tsudo, M., et al., 1984, J. Exp. Med. 160:612-617; Waldmann, T.A., et al., 1984, J. Exp. Med. 160:1450-1466).

Interaction of IL-2 with its cell surface receptor results in acontinuous T cell proliferation (Greene, W. C. and Leonard, W. J., 1986,Ann. Rev. Immunol. 4:69-95; Smith, K. A., 1984, Ann. Rev. Immunol.2:319-333). Measurement of IL-2r provides information on the state ofimmune activation of the lymphoid population. This has been accomplishedby measuring IL-2r on cell surfaces using flow cytometry or fluorescencemicroscopy. Using monoclonal antibodies which define the IL-2 receptor,altered IL-2r expression has been reported in a number of immuneabnormalities (Greene and Leonard, supra; Depper, J. M., et al., 1984,J. Immunol. 133:1691-1695) such as certain B- or T-cell malignancies,including Burkitt's lymphoma (Waldmann, T. A., et al., 1984, J. Exp.Med. 160:1450-1466), hairy cell leukemia (Waldmann et al., supra;Korsmeyer, S. J., et al., 1983, Proc. Natl. Acad. Sci. U.S.A.80:4522-4526, and human T cell leukemia virus (HTLV)-I-associated adultT cell leukemia (Depper, J. M., et al., 1984, J. Immunol. 133:1691-1695). In several cases of common, pre-B or T cell acute lymphoblasticleukemia (ALL), malignant cells have been induced to express IL-2r afterin vitro activation (Touw, I., et al., 1985, Blood 66:556-561; Touw, I.,et al., 1986, Blood 68:1088-1094; Matsuoka, M., et al., 1986, Leuk. Res.10:597-603) and, in some cases, interleukin-2 stimulated subsequentcolony formation of neoplastic progenitor cells in vitro (Touw, 1985,supra; Touw, 1986, supra).

Leukemic cells from some patients with T cell chronic lymphocyticleukemia were shown to have the receptors and a good proliferativeresponse to exogenous interleukin-2 (Uchiyama, T., et al., 1985, J.Clin. Invest. 76:446-453; Tsudo, M., 1986, Blood 67:316-321). However,HTLV-1 associated adult T cell leukemic cells constitutively expressedhigh levels of cell surface IL-2r but had no or very poor proliferativeresponses to interleukin-2 (Uchiyama, 1985, supra; Arya, S. K., et al.,1984, Science 223:1086-1087). Ebert et al. (1985, Clin. Immunol.Immunopathol. 37:283-297) have reported that T cells from patients withAIDS virus lack the ability to express IL-2r on their surface even whenthe cell is activated.

Secondary signals in T cell activation such as interleukin-1 areprovided by monocytes or other accessory cells, and are required forIL-2 secretion (Schmidtke, J. R. and Hatfield, S., 1976, J. Immunol.116:357; Maizel, A. L., et al., 1981, J. Exp. Med. 153:470; Williams, J.M., et al., 1985, J. Immunol. 135:2249).

2.3. Human Immunodeficiency Virus Type I And Other Human Retroviruses

Retroviruses are enveloped RNA tumor viruses (for a review, see Hayward,W. S. and Neel, B. G., 1981, Curr. Top. Microbiol. Immunol. 91:217-276).The virus particle consists of a ribonucleoprotein core enclosed by anouter membrane envelope derived from the host cell plasma membrane.Viral envelope glycoproteins protrude from the outer envelope. The viralgenome consists of a single-stranded RNA molecule.

The long terminal repeat (LTR) region of retroviruses is found at theends of the proviral DNA, and consists of three defined segments: U3(derived from the 3' end of genomic RNA, R (a short terminal repeat ofgenomic RNA), and U5 (derived from the 5' end of genomic RNA). Thepromoter for retroviral RNA synthesis appears to be located within theLTR, in the U3 region (id.). The LTR regions of several HTLV isolateshave been analyzed (Sodroski, J., et al., 1984, in Human T-CellLeukemia/Lymphoma Virus, Gallo, R. C., et al., eds., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., pp. 149-155).

HTLV-I and HTLV-II are related but distinct human retrovirusesassociated with certain leukemias and lymphomas. HTLV-I is causativelylinked to adult T cell leukemia (Gallo, R. C. and Wong-Staal, F., 1982,Blood 60:545; Gallo, R. C., 1984, in Cancer Surveys, Vol. 3, Franks, L.M., et al., eds., Oxford Univ. Press, Oxford, pp. 113-159). HTLV-II wasfirst identified in a patient with a T cell variant of hairy cellleukemia (Kalyanaraman, V. S., et al., 1982, Science 218:571). Bothviruses show a tropism for human T cells, and have the capacity totransform infected T cells in vitro, in addition to causing othercellular changes (see Arya, S. K., et al., 1984, Science 225:927-930,and references cited therein).

The causative agent of AIDS is a retrovirus, now termed humanimmunodeficiency virus type 1 (HIV-1), and formerly termed HTLV III(Gallo, R. C., et al., 1984, Science 224:500, Popovic, M., et al., 1984,Science 224:497), LAV (Barre-Sinoussi, F., et al., 1983, Science220:868; Feorino, P. M., et al., 1984, Science 225:69), and ARV (Levy,J. A., et al., 1984, Science 225:840) by the three groups whichindependently isolated viruses which are probably of the same retrovirussubgroup (Levy, J. A., et al., 1984, Science 225:840). HIV-1 appears toinfect T4⁺ helper T lymphocytes, and evidence suggests that the T4antigen is the receptor or a component of the receptor for the virus(Dagleish, A. G., et al., 1984, Nature 312:763; Klatzmann, D., et al.,1984, Nature 312:767).

The structure of the RNA genome of HIV-1 has been described (Ratner, L.,et al., 1985, Nature 313:277-284) and includes the gag (group-specificantigens; encoding internal structural proteins), pol (encoding thereverse transcriptase), env (encoding the envelope glycoproteins), sor(short open reading frame), 3'-orf (Guy, B., et al., 1987, Nature330:266-269), art/trs, and tat (Kao, S.-Y., et al., 1987, Nature330:489-493) genes.

AIDS is a disease which is characterized by a severe immune deficiencyprimarily caused by a decreased cell-mediated immune response (Gottlieb,M., et al., 1981, N. Engl. J. Med. 305:1425; Masur, J., et al., 1981, N.Engl. J Med. 305:1431). The immunodeficient state is characterized by adecrease in T_(H) (T helper) lymphocytes, a reversal of the normal T4⁺:T8⁺ cell ratio, lymphopenia, and opportunistic infections often causedby Pneumocystis carinii. Some patients also develop lymphoma or Kaposi'ssarcoma at increased incidence. The disease is usually fatal.

3. SUMMARY OF THE INVENTION

The present invention is directed to genes, termed Rpt-1 (regulatoryprotein T lymphocyte-1), which are expressed at higher levels by restingCD4⁺ helper/inducer T cells relative to activated CD4⁺ cells. Theinvention also relates to the proteins encoded by such genes, termedrpt-1 proteins, which regulate gene expression directed by the promoterregion of the interleukin-2 receptor (IL-2r) alpha chain gene or by thepromoter region of the long terminal repeat of human lymphotropicretroviruses such as human immunodeficiency virus type-1 (HIV-1), humanT cell leukemia virus (HTLV)-I, and HTLV-II. In particular, rpt-1proteins down-regulate gene expression controlled by the promoter of theIL-2r alpha chain gene or by the promoter of the long terminal repeat ofHIV-1 The proteins and nucleic acids of the invention have value indiagnosis and therapy of immune disorders such as AIDS.

In a specific example of the present invention detailed infra, an Rpt-1gene and its encoded intracellular protein of approximately 41,000daltons molecular weight are described. The rpt-1 protein is shown to beselectively expressed by activated CD4⁺ T cells, and to down-regulategene expression of the IL-2r and the HIV-1.

    ______________________________________                                                 3.1. DEFINITIONS                                                     ______________________________________                                        bp         =     base pair                                                    CAT        =     chloramphenicol acetyl                                                        transferase                                                  ConA       =     concanavalin A                                               FACS       =     fluorescence activated cell                                                   sorter                                                       FITC       =     fluorescein isothiocyanate                                   HBB        =     hemoglobin b chain                                           HIV        =     human immunodeficiency virus                                 HTLV       =     human T cell leukemia virus                                  IL         =     interleukin                                                  IL-2r      =     interleukin-2 receptor                                       kb         =     kilobase pair                                                KLH        =     keyhole limpet hemocyanin                                    LTR        =     long terminal repeat                                         PBS        =     phosphate-buffered saline                                    PHA        =     phytohemagglutinin                                           RFLP       =     restriction fragment length                                                   polymorphism                                                 Rpt-1      =     regulatory protein                                                            T-lymphocyte-1                                               TPA        =     12-0-tetradecanoylphorbol                                                     13-acetate                                                   TSA        =     Tris saline azide                                            ______________________________________                                    

4. DESCRIPTION OF THE FIGURES

FIG. 1A. Expression of Rpt-1 in lymphocyte clones. 5 ug of poly(A)⁺ RNAfrom each cell type were analyzed by electrophoresis on a 1.5% agarosegel, transferred to nitrocellulose, and hybridized with a ³² P-labeledprobe prepared by nick translation of the 3.7 kb cDNA insert ofpcD-rpt1. Cell types: MOPC 315, a murine myeloma (lane a); Cl.NK1.1, aThy 1⁺ natural killer cell clone (lane b); Cl.Ly23.4, a suppressor Tcell clone with (lane c) or without (lane d) ConA; Cl.Ly1-T1, a T-helperclone without (lane e) and with (lane f) ConA; and Cl.Ly1-N5, a T-helperclone without (lane g) and with (lane h) ConA. RNA was obtained 15 hoursafter activation. As determined by densitometric scanning, the intensityof the radioactive signal in lanes f and h is approximately 50% of thatin lanes e and g, respectively, even when the levels of actin mRNA areused as internal controls for normalization. Molecular weight markerscorrespond to: 6000, 1765, 1426, and 920 bases.

FIG. 1B. Expression of Rpt-1 in heterogeneous lymphocyte populations. 5ug of poly(A)⁺ RNA from splenocytes (lane a) or thymocytes (lane b) washybridized with the Rpt-1 insert as described above.

FIG. 1C. Time course of Rpt-1 expression upon activation of the T cellclone Cl.Ly1-T1.5 ug of total RNA was obtained from Cl.Ly1-T1 at theindicated times (hours) after stimulation by antigen (TNP-BGG;trinitrophenyl-bovine gamma-globulin) and splenic adherent cells.Northern blots were hybridized with a nick-translated probecorresponding to the 5' 1.3 kb RsaI-XbaI fragment (coding region) ofRpt-1 (upper panel) and to gamma-interferon (lower panel).

FIG. 2. Restriction map of the insert of pcD-rpt1 and sequence of thecoding region. The sequence was determined by the method of Maxam andGilbert (1977, Proc. Natl. Acad. Sci. U.S.A. 74:560-564) and wasconfirmed in its entirety on both strands. The restriction map includesarrows to indicate the extent and direction of sequence determined, andasterisks to denote potential polyadenylation sites. TCR7-D and TCR7-F2are partial cDNA clones and TCR7-X is a full-length cDNA clone. Cysteineand histidine residues in the predicted amino acid sequence that may beinvolved in metal finger formation are circled. The first methionine,the predicted most hydrophilic region (amino acid residue numbers205-210) and putative nuclear localization signal (amino acid numbers268-276) are boxed.

FIG. 3. Hydrophilicity plot of the predicted rpt-1 protein (Hopp, T. andWoods, K., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828). Above thehorizontal line, hydrophilic; below, hydrophobic. Potential fingerstructures including the pairs of cysteine and histidine residues markedabove the plot (circled in FIG. 2) are schematized in the lower panel(Me: metal). The location of the predicted most hydrophilic region inrpt-1 (KKEKKE) is shown (black box) as well as the subsequence similarto the nuclear localization signal of the SV40 large T antigen (NL)(Sabatini, D.D., et al., 1982, J. Cell. Biol. 92:1-19).

FIG. 4A. Western blot of mourse spleen cell extracts using anti-KKEKKEantibody. Molecular weight markers (Sigma Chemical Co.) correspond to:58,000; 48,500; and 36,500 daltons.

FIG. 4B. COS7m6 cells were transfected with 6 ug of either pcD-rpt1(COS-rpt1⁺) or pcD-rpt1fs (COS-rpt1fs). The FACS fluorescence profile ofan aliquot of each cell population after incubation with the anti-KKEKKEantibody is shown. Slot blot analysis of RNA from both groups of COS7m6cells revealed equal amounts of mRNA that hybridized to the Rpt-1 probe.

FIG. 4C. Immunofluorescence of both cell populations was also examinedusing a fluorescence microscope. COS-rpt-1 transfectants showedpredominantly nuclear fluorescence as illustrated in thephotomicrograph. Fluorescence was not detectable in the COS-rpt-1fscells.

FIG. 5. Southern blot analysis of liver. Genomic DNA from C57B1/6 (B),DBA (D), Cl.Ly1-T1 (BALB/c), and the numbered recombinant inbred strainsderived from B and D parental strains (BXD) (Jackson Laboratories, BarHarbor, Me.) (Taylor, B. A., 1981, in Genetic Variants and Strains ofthe Laboratory Mouse, Academic Press, New York, pp. 397-407) weredigested with HindIII. The genomic DNA was analyzed in a 0.7% agarosegel, and blotted onto a Zetaprobe membrane (BioRad, Richmond, Calif.).The filter was hybridized with a ³² P-labelled probe prepared by nicktranslation of the 5' 1.3 kb RsaI-XbaI fragment (coding region) ofpcD-rpt1.

FIG. 6A. Indirect immunofluorescent staining and FACS analysis of rpt-1and IL-2rα expression by clones Ar-5 (left panel) and Ar-5v (centerpanel). IL-2rα fluorescence of Ar-5 was at background levels comparableto that obtained using a control antibody. Twenty-four hours afterstimulation with recombinant IL-2, aliquots of Ar-5v were transfectedwith 6 ug of pcD, pcD-rpt1fs, or pcD-rpt1 48 hours before measurement ofsurface I1-2rα by FACS analysis (right panel). Approximately one-thirdof the cells transfected with pcD-rpt1 (hatched bars), but not cellstransfected with pcD-rpt1fs, no longer expressed significant levels ofIL-2rα (compare to IL-2rα FACS profile of Ar-5, left panel). The levelsof IL-2rα expressed by cells transfected with pcD-rpt1fs (shown in theright panel) did not differ significantly from those of cellstransfected with pcD or with pcD-beta-galactosidase. Slot blot analysisof 250 ug total RNA showed that cells transfected with pcD-rpt1 andpcD-rpt1 fs, but not pcD (or pcD-beta-galactosidase), contained highlevels of Rpt-1 mRNA. All of these transfectants had similar levels ofIL-3 mRNA.

FIG. 6B. Chloramphenicol acetyl transferase (CAT) assays of COS7m6, andresting and activated EL-4 and Jurkat cells. T cells were activated 20hours before the CAT assays. CAT reaction mixtures were incubated for 20minutes. Results are shown after transfection with the followingplasmids: COS7m6 cells: 4 ug of IL2rpCAT (plasmid containing the IL-2rαpromoter upstream of the bacterial chloramphenicol gene [CAT])co-transfected with 6 ug of either pcD-rpt1 (lane 1) or pcD-rpt1fs (lane2); 3 ug of pLTR-1CAT (plasmid containing the HIV-1 LTR upstream ofCAT), co-transfected with 6 ug of either pcD-rpt1 (lane 3) or pcD-rpt1fs(lane 4); 2 ug of pSV2CAT co-transfected with 6 ug of either pcD-rpt1(lane 5) or pcD-rpt1fs (lane 6). EL-4 cells: 5 ug of pLTR-1CAT and 4 ugof pSV 7fdtat cotransfected alone (lanes 7, 10), or with 6 ug of eitherpcD-rpt1 (lanes 8, 11) or pcD-rpt1fs (lanes 9, 12). Jurkat cells: 5 ugof pLTR-1CAT and 2 ug of pSV7fdtat co-transfected alone (lanes 13, 16)or with 3 ug of either pcD-rpt1 (lanes 14, 17) or pcD-rpt1fs (lanes 15,18). The inhibitory effect of rpt-1 was also seen when using half (3 ug)and twice (9 ug) the amount of pcD-rpt1, and it did not depend on theamount of indicator and poD-rpt1 plasmids used as long as the ratio oftheir amounts remained the same as above. Cotransfections that includedthe pcD vector displayed lower CAT activity levels due to competitionbetween promoters for transcriptional factors. The chloramphenicolconversion (%) after co-transfection with pcD-rpt1 is expressed as aproportion of the chloramphenicol conversion (%) obtained afterco-transfection with pcD-rpt1fs with the same CAT plasmid.Chloramphenicol conversion (%) obtained with pcD-rpt1fs cotransfectionswere assigned a value of 1. Lanes 7, 10, 13, and 16 are included ascontrols for T cell activation. Extracts from cells that had not beentransfected with CAT plasmids showed no CAT activity. Each experimentwas repeated three to five times. Standard errors of absolutechloramphenicol conversion percentages obtained in each experiment wereless than 30% of the mean.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to genes, termed Rpt-1 (regulatoryprotein T lymphocyte-1), which are expressed at higher levels by restingCD4⁺ helper/inducer T cells relative to activated CD4⁺ cells. Theinvention also relates to the proteins encoded by such genes, termedrpt-1 proteins, which regulate gene expression directed by the promoterregion of the interleukin-2 receptor (IL-2r) alpha chain gene or by thepromoter region of the long terminal repeat (LTR) of human lymphotropicretroviruses such as human immunodeficiency virus type 1 (HIV-1), humanT cell leukemia virus (HTLV)-I, and HTLV-II. In particular, rpt-1proteins down-regulate gene expression controlled by the promoter of theIL-2r alpha chain gene or by the promoter of the LTR of HIV-1. Theproteins and nucleic acids of the invention have value in diagnosis andtherapy of immune disorders such as AIDS.

In a specific example of the present invention detailed infra, an Rpt-1gene and its encoded intracellular protein of approximately 41,000daltons molecular weight are described. The rpt-1 protein is shown to beselectively expressed by activated CD4⁺ T cells, and to down-regulategene expression of the IL-2r and the HIV-1.

5.1 Isolation of the Rpt-1 Gene

Any mammalian cell can potentially serve as the nucleic acid source forthe molecular cloning of the Rpt-1 gene. Isolation of the Rpt-1 geneinvolves the isolation of those DNA sequences which encode a proteindisplaying Rpt-1-associated structure or properties, e.g.,down-regulation of gene expression of the IL-2r or the HIV-1 (seeSection 5.5, infra). The DNA may be obtained by standard proceduresknown in the art from cloned DNA (e.g., a DNA "library"), by chemicalsynthesis, by cDNA cloning, or by the cloning of genomic DNA, orfragments thereof, purified from the desired mammalian cell. (See, forexample, Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Glover, D. M.(ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford,U.K., Vol. I, II.) Clones derived from genomic DNA may containregulatory and intron DNA regions in addition to coding regions; clonesderived from cDNA will contain only exon sequences. Whatever the source,the Rpt-1 gene should be molecularly cloned into a suitable vector forpropagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired Rpt-1 gene. The DNA maybe cleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the Rpt-1 gene may be accomplished in a number ofways. For example, if an amount of an Rpt-1 gene or its specific RNA, ora fragment thereof, is available and can be purified and labeled, thegenerated DNA fragments may be screened by nucleic acid hybridization tothe labeled probe (Benton, W. and Davis, R., 1977, Science 196:180;Grunstein, M. and Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A.72:3961). Those DNA fragments with substantial homology to the probewill hybridize. If a purified Rpt-1-specific probe is unavailable,nucleic acid fractions enriched in Rpt-1 may be used as a probe, as aninitial selection procedure. As an example (see Section 6.1.3, infra),the probe representing T cell cDNA from which messages expressed byactivated T cells have been subtracted can be used. It is also possibleto identify the appropriate fragment by restriction enzyme digestion(s)and comparison of fragment sizes with those expected according to aknown restriction map if such is available. Further selection on thebasis of the properties of the gene, or the physical, chemical, orimmunological properties of its expressed product, as described infra,can be employed after the initial selection.

The Rpt-1 gene can also be identified by mRNA selection by nucleic acidhybridization followed by in vitro translation. In this procedure,fragments are used to isolate complementary mRNAs by hybridization. SuchDNA fragments may represent available, purified Rpt-1 DNA, or DNA thathas been enriched for Rpt-1 sequences (e.g., cDNA enriched for messagesof resting T cells). Immunoprecipitation analysis or functional assays(e.g., for IL-2r or HIV-1 promoter regulation) of the in vitrotranslation products of the isolated mRNAs identifies the mRNA and,therefore, the complementary DNA fragments that contain the Rpt-1sequences. In addition, specific mRNAs may be selected by adsorption ofpolysomes isolated from cells to immobilized antibodies specificallydirected against an rpt-1 protein. A radiolabeled Rpt-1 cDNA can besynthesized using the selected mRNA (from the adsorbed polysomes) as atemplate. The radiolabeled mRNA or cDNA may then be used as a probe toidentify the Rpt-1 DNA fragments from among other genomic DNA fragments.

Alternatives to isolating the Rpt-1 genomic DNA include, but are notlimited to, chemically synthesizing the gene sequence itself from aknown sequence or making cDNA to the mRNA which encodes the Rpt-1 gene.For example, RNA for cDNA cloning of the Rpt-1 gene can be isolated fromcells including but not limited to immune cells such as T cells. Othermethods are possible and within the scope of the invention.

The identified and isolated gene can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas PBR322 or pUC plasmid or pcD/Okayama-Berg plasmid (Okayama, H. andBerg, P., 1983, Mol. Cell. Biol. 3:280-289) derivatives. Recombinantmolecules can be introduced into host cells via transformation,transfection, infection, electroporation, etc.

In a particular embodiment, the Rpt-1 gene expressed in resting T cellscan be cloned by selection from a constructed sublibrary that containscDNA inserts that are selectively expressed by CD4⁺ T helper/inducercells. Such a procedure is described in Sections 6.1.1 through 6.1.4,infra.

In an alternative method, the Rpt-1 gene may be identified and isolatedafter insertion into a suitable cloning vector, in a "shot gun" approachEnrichment for the Rpt-1 gene, for example, by size fractionation, canbe done before insertion into the cloning vector.

The Rpt-1 gene is inserted into a cloning vector which can be used totransform, transfect, or infect appropriate host cells so that manycopies of the gene sequences are generated. In a specific embodiment,the cloning vector can be the pcD vector (Okayama-Berg vector; Okayama,H. and Berg, P., 1983, Mol. Cell. Biol. 3:280-289). The insertion into acloning vector can, for example, be accomplished by ligating the DNAfragment into a cloning vector which has complementary cohesive termini.However, if the complementary restriction sites used to fragment the DNAare not present in the cloning vector, the ends of the DNA molecules maybe enzymatically modified. Alternatively, any site desired may beproduced by ligating nucleotide sequences (linkers) onto the DNAtermini; these ligated linkers may comprise specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. In an alternative method, the cleaved vector andRpt-1 gene may be modified by homopolymeric tailing.

Identification of the cloned Rpt-1 gene can be accomplished in a numberof ways based on the properties of the DNA itself, or alternatively, onthe physical, immunological, or functional properties of its encodedprotein. For example, the DNA itself may be detected by plaque or colonynucleic acid hybridization to labeled probes (Benton, W. and Davis, R.,1977, Science 196:180; Grunstein, M. and Hogness, D., 1975, Proc. Natl.Acad. Sci. U.S.A. 72:3961). Alternatively, the presence of the Rpt-1gene may be detected by assays based on properties of its expressedproduct. For example, cDNA clones, or DNA clones which hybrid-select theproper mRNAs, can be selected which produce a protein that, e.g., hassimilar or identical electrophoretic migration, isoelectric focusingbehavior, proteolytic digestion maps, down-regulation of IL-2r or HIV-1promoter activity, or antigenic properties as known for rpt-1. If anantibody to rpt-1 is available, the rpt-1 protein may be identified bybinding of labeled antibody to the putatively rpt-1-synthesizing clones,in an ELISA (enzyme-linked immunosorbent assay)-type procedure.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated Rpt-1 gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene may be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

In a particular embodiment, Rpt-1 cDNA clones in a pcD vector (Okayama,H. and Berg, P., 1983, Mol. Cell. Biol. 3:280-289) can be transfectedinto COS (monkey kidney) cells for large-scale expression (see Section6.1.8, infra).

If the ultimate goal is to insert the gene into virus expression vectorssuch as vaccinia virus or adenovirus, the recombinant DNA molecule thatincorporates the Rpt-1 gene can be modified so that the gene is flankedby virus sequences that allow for genetic recombination in cellsinfected with the virus so that the gene can be inserted into the viralgenome.

After the Rpt-1 DNA-containing clone has been identified, grown, andharvested, its DNA insert may be characterized as described in Section5.4.1, infra.

When the genetic structure of the Rpt-1 gene is known, it is possible tomanipulate the structure for optimal use in the present invention. Forexample, promoter DNA may be ligated 5' of the rpt-1-coding sequence, inaddition to or replacement of the native promoter to provide forincreased expression of the protein. Many manipulations are possible,and within the scope of the present invention.

5.2. Expression of the Rpt-1 Gene

The nucleotide sequence coding for an rpt-1 protein or a portionthereof, can be inserted into an appropriate expression vector, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted protein-coding sequence. The necessarytranscriptional and translation signals can also be supplied by thenative Rpt-1 gene and/or its flanking regions. A variety of host-vectorsystems may be utilized to express the protein-coding sequence. Theseinclude but are not limited to mammalian cell systems infected withvirus (e.g., vaccinia virus, adenovirus, etc.); insect cell systemsinfected with virus (e.g., baculovirus); microorganisms such as yeastcontaining yeast vectors, or bacteria transformed with bacteriophageDNA, plasmid DNA or cosmid DNA. The expression elements of these vectorsvary in their strength and specificities. Depending on the host-vectorsystem utilized, any one of a number of suitable transcription andtranslation elements may be used. For instance, when cloning inmammalian cell systems, promoters isolated from the genome of mammaliancells or from viruses that grow in these cells may be used. Promotersproduced by recombinant DNA or synthetic techniques may also be used toprovide for transcription of the inserted sequences.

Specific initiation signals are also required for efficient translationof inserted protein coding sequences. These signals include the ATGinitiation codon and adjacent sequences. In cases where the entire Rpt-1gene including its own initiation codon and adjacent sequences areinserted into the appropriate expression vectors, no additionaltranslational control signals may be needed. However, in cases whereonly a portion of the Rpt-1 coding sequence is inserted, exogenoustranslational control signals, including the ATG initiation codon, mustbe provided. The initiation codon must furthermore be in phase with thereading frame of the protein coding sequences to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic.

Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a chimeric gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinations (genetic recombination).

Expression vectors containing Rpt-1 gene inserts can be identified bythree general approaches: (a) DNA-DNA hybridization, (b) presence orabsence of "marker" gene functions, and (c) expression of insertedsequences. In the first approach, the presence of a foreign geneinserted in an expression vector can be detected by DNA-DNAhybridization using probes comprising sequences that are homologous tothe inserted Rpt-1 gene. In the second approach, the recombinantvector/host system can be identified and selected based upon thepresence or absence of certain "marker" gene gunctions (e.g., thymidinekinase activity, resistance to antibiotics, transformation phenotype,occlusion body formation in baculovirus, etc.) caused by the insertionof foreign genes into the vector. For example, if the Rpt-1 gene isinserted within the marker gene sequence of the vector, recombinantscontaining the Rpt-1 insert can be identified by the absence of themarker gene function. In the third approach, recombinant expressionvectors can be identified by assaying the foreign gene product expressedby the recombinant. Such assays can be based on the physical,immunological, or functional properties of the gene product.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity.

In a particular embodiment detailed in the examples of the presentinvention, pcD vectors with an Rpt-1 cDNA insert can be transfected intoCOS cells, in which the Rpt-1 cDNA insert is expressed to produce therpt-1 protein. However, the invention is not limited to the expressionof Rpt-1 from pcD vectors in COS cells. As previously explained, theexpression vectors which can be used include, but are not limited to,the following vectors or their derivatives: human or animal viruses suchas vaccinia virus or adenovirus; insect viruses such as baculovirus;yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid andcosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes thechimeric gene product in the specific fashion desired. Expression fromcertain promoters can be elevated in the presence of certain inducers;thus, expression of the genetically engineered rpt-1 protein may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the desired modification and processingof the expressed heterologous protein. For example, in one embodiment,expression in a bacterial system can be used to produce the 41.3 kdrpt-1 protein with the deduced amino acid sequence of FIG. 2. In anotherembodiment, mammalian COS cells can be used to ensure "native"conformation of the heterologous rpt-1 protein. Furthermore, differentvector/host expression systems may effect processing reactions such asproteolytic cleavages to different extents. Many such variouslyprocessed rpt-1 proteins can be produced and are within the scope of thepresent invention.

5 3. Identification and Purification of the Expressed Gene Product

Once a recombinant which expresses the Rpt-1 gene is identified, thegene product should be analyzed. This can be achieved by assays based onthe physical, immunological, or functional properties of the product.

Once the rpt-1 protein is identified, it may be isolated and purified bystandard methods including chromatography (e.g., ion exchange, affinity,and sizing column chromatography), centrifugation, differentialsolubility, or by any other standard technique for the purification ofproteins.

Alternatively, once an rpt-1 protein produced by a recombinant isidentified, the amino acid sequence of the protein can be deduced fromthe nucleotide sequence of the chimeric gene contained in therecombinant. As a result, the protein can be synthesized by standardchemical methods known in the art (e.g., see Hunkapiller, M., et al.,1984, Nature 310:105-111).

In particular embodiments of the present invention, such rpt-1 proteins,whether produced by recombinant DNA techniques or by chemical syntheticmethods, include but are not limited to those containing, as a primaryamino acid sequence, all or part of the amino acid sequencesubstantially as depicted in FIG. 2, including altered sequences inwhich functionally equivalent amino acid residues are substituted forresidues within the sequence resulting in a silent change. For example,one or more amino acid residues within the sequence can be substitutedby another amino acid of a similar polarity which acts as a functionalequivalent, resulting in a silent alteration. Substitutes for an aminoacid within the sequence may be selected from other members of the classto which the amino acid belongs. For example, the nonpolar (hydrophobic)amino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Also included within the scopeof the invention are rpt-1 proteins which are differentially modifiedduring or after translation, e.g., by glycosylation, proteolyticcleavage, etc.

5.4. Structure of the Rpt-1 Gene and Protein

The structure of the Rpt-1 gene and protein can be analyzed by variousmethods known in the art.

5.4.1. Genetic Analysis

The cloned DNA or cDNA corresponding to the Rpt-1 gene can be analyzedby methods including but not limited to Southern hybridization(Southern, E. M., 1975, J. Mol. Biol. 98:503-517), Northernhybridization (see e.g., Freeman et al., 1983, Proc. Natl. Acad. Sci.U.S.A. 80:4094-4098), restriction endonuclease mapping (Maniatis, T.,1982, Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.), and DNA sequence analysis.Southern hybridization with an Rpt-1-specific probe can allow thedetection of the Rpt-1 gene in various cell types. In one embodiment,Southern hybridization can be used to determine the genetic linkage ofRpt-1 (see Section 6.2). Northern hybridization analysis can be used todetermine the expression of the Rpt-1 gene. Various cell types, atvarious states of development or activity can be tested for Rpt-1expression. Such a technique and its results, demonstrating Rpt-1expression by inducer T cells, but not by suppressor or cytolytic Tcells, or by natural killer cells, are described in Sections 6.1.5 and6.2, infra. The stringency of the hybridization conditions for bothSouthern and Northern hybridization can be manipulated to ensuredetection of nucleic acids with the desired degree of relatedness to thespecific Rpt-1 probe used.

Restriction endonuclease mapping can be used to roughly determine thegenetic structure of the Rpt-1 gene. In a particular embodiment,cleavage with restriction enzymes can be used to derive the restrictionmap shown in FIG. 2, infra. Restriction maps derived by restrictionendonuclease cleavage can be confirmed by DNA sequence analysis.

DNA sequence analysis can be performed by any techniques known in theart, including but not limited to the method of Maxam and Gilbert (1980,Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger, F., etal., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463), or use of anautomated DNA sequenator (e.g., Applied Biosystems, Foster City,Calif.). The cDNA sequence of a representative Rpt-1 gene comprises thesequence substantially as depicted in FIG. 2, and described in Section6.2, infra.

5.4.2. Protein Analysis

The amino acid sequence of the rpt-1 protein can be derived by deductionfrom the DNA sequence, or alternatively, by direct sequencing of theprotein, e.g., with an automated amino acid sequencer. The amino acidsequence of a representative rpt-1 protein comprises the sequencesubstantially as depicted in FIG. 2, and detailed in Section 6.2, infra.

The rpt-1 protein sequence can be further characterized by ahydrophilicity analysis (Hopp, T. and Woods, K., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:3824). A hydrophilicity profile can be used to identifythe hydrophobic and hydrophilic regions of the rpt-1 protein and thecorresponding regions of the gene sequence which encode such regions. Ahydrophilicity profile of the rpt-1 protein described in the examplessection infra is depicted in FIG. 3.

Secondary structural analysis (Chou, P. and Fasman, G., 1974,Biochemistry 13:222) can also be done, to identify regions of rpt-1 thatassume specific secondary structures.

Other methods of structural analysis can also be employed. These includebut are not limited to X-ray crystallography (Engstom, A., 1974,Biochem. Exp. Biol. 11:7-13) and computer modeling (Fletterick, R. andZoller, M. (eds.), 1986, Computer Graphics and Molecular Modeling, inCurrent Communications in Molecular Biology, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

5.5. Regulation of Gene Expression of the Interleukin-2 Receptor and ofHuman Lymphotropic Retroviruses

The rpt-1 proteins function to regulate the level of gene expressiondirected by the promoters of the interleukin-2 receptor or of the LTRsof human lymphotropic retroviruses such as HIV-1, HTLV-I, and HTLV-II(see Sections 6.2 and 6.3, infra). Expression of the Rpt-1 gene resultsin the decreased expression of genes whose transcription is under thecontrol of the IL-2r promoter (in particular, that of the IL-2rα chain),or under the control of the HIV-1 LTR promoter. It is envisioned thatthe expression of genes under the control of promoters such as that ofthe IL-2rβ chain or HIV type 2 LTR or the LTR of similar retroviruses,can also be regulated by the rpt-1 proteins of the invention.

5.6. Anti-Rpt-1 Antibody Production

Antibodies can be produced which recognize the rpt-1 protein. Suchantibodies can be polyclonal or monoclonal.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to rpt-1. In a particular embodiment, rabbitpolyclonal antibodies to an epitope of the rpt-1 molecule depicted inFIG. 2 can be obtained as described in Section 6.1.6, infra. For theproduction of antibody, various host animals can be immunized byinjection with the native rpt-1 protein, or a synthetic version, orfragment thereof, including but not limited to rabbits, mice, rats, etc.Various adjuvants may be used to increase the immunological response,depending on the host species, and including but not limited to Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and corynebacterium parvum.

A monoclonal antibody to rpt-1 can be prepared by using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include but are not limited to thehybridoma technique originally described by Kohler and Milstein (1975,Nature 256:495-497), and the more recent human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72) andEBV-transformation technique (Cole et al., 1985, Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab')₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab' fragments which can begenerated by reducing the disulfide bridges of the F(ab')₂ fragment, andthe 2 Fab or Fab fragments which can be generated by treating theantibody molecule with papain and a reducing agent.

5.7. Rpt-1-Related Derivatives, Analogues, and Peptides

The production and use of derivatives, analogues, and peptides relatedto rpt-1 are also envisioned, and within the scope of the presentinvention. Such derivatives, analogues, or peptides which have thedesired immunogenicity or antigenicity can be used, for example, inimmunoassays, for immunization, therapeutically, etc. Such moleculeswhich retain, or alternatively inhibit, a desired rpt-1 property, e.g.,down-regulation of IL-2r or HIV promoter-directed gene expression, canbe used as inducers, or inhibitors, respectively, of such property.Derivatives, analogues, or peptides related to rpt-1 can be tested forthe desired activity by procedures known in the art, including but notlimited to the assays described in Sections 5.5 and 6.1.8.

The rpt-1-related derivatives, analogues, and peptides of the inventioncan be produced by various methods known in the art. The manipulationswhich result in their production can occur at the gene or protein level.For example, the cloned Rpt-1 gene can be modified by any of numerousstrategies known in the art (Maniatis, T., 1982, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.). The Rpt-1 sequence can be cleaved at appropriate sites withrestriction endonuclease(s), followed by further enzymatic modificationif desired, isolated, and ligated in vitro. In the production of thegene encoding a derivative, analogue, or peptide related to rpt-1, careshould be taken to ensure that the modified gene remains within the sametranslational reading frame as rpt-1, uninterrupted by translationalstop signals, in the gene region where the desired rpt-1-specificactivity is encoded.

Additionally, the Rpt-1 gene can be mutated in vitro or in vivo, tocreate and/or destroy translation, initiation, and/or terminationsequences, or to create variations in coding regions and/or form newrestriction endonuclease sites or destroy preexisting ones, tofacilitate further in vitro modification. Any technique for mutagenesisknown in the art can be used, including but not limited to, in vitrosite-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem.253:6551), use of TAB® linkers (Pharmacia), etc.

Manipulations of the rpt-1 sequence may also be made at the proteinlevel. Any of numerous chemical modifications may be carried out byknown techniques, including but not limited to specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄ ; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

In addition, analogues and peptides related to rpt-1 can be chemicallysynthesized. For example, a peptide corresponding to a portion of rpt-1which mediates the desired regulation of gene expression can besynthesized by use of a DNA synthesizer (e.g., Applied Biosystems Model380A).

5.8. Uses of Rpt-1 5.8.1. Diagnosis

rpt-1 proteins, analogues, derivatives, and subsequences thereof, andanti-rpt-1 antibodies, have uses in diagnostics. The molecules of theinvention can be used in assays, such as immunoassays, to detect,prognose, diagnose, or monitor various conditions, diseases, anddisorders affecting T lymphocyte activation. For example, the amount ofrpt-1 expressed in T cells appears to be inversely proportional to theamount of IL-2r present on the cell-surface (see Section 6.2 infra, FIG.6A), which is a measure of T cell activation. Thus, in a specificembodiment, antibody to rpt-1 can be used to assay in a patient tissueor serum sample for the presence of rpt-1, where a decreased level ofrpt-1 is an indicator of increased IL-2r cell-surface expression and Tcell (immune) activation. In another embodiment, the detection ofincreased expression of rpt-1 can be viewed as a signal of decreasedIL-2r cell-surface expression, which may be indicative of poor T cellresponses and relative T cell resistance to activation. It is envisionedthat various immune abnormalities such as congenital and acquired immunedeficiencies, autoimmune disorders (e.g., systemic lupus erythematosus,rheumatoid arthritis, and diabetes type I), and various cancers may besusceptible to classification according to the levels found in patientsamples of various immune function mediators, including rpt-1. Suchclassifications can be of great value in the prognosis or diagnosis ofimmune abnormalities.

The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"immunoassays, precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays, and immunoelectrophoresis assays, to name but a few.

Rpt-1 genes and related nucleic acid sequences and subsequences,including complementary sequences, can also be used in hybridizationassays. The Rpt-1 nucleic acid sequences, or subsequences thereofcomprising about at least 15 nucleotides, can be used as hybridizationprobes. Hybridization assays can be used to detect, prognose, diagnose,or monitor conditions, disorders, or disease states associated withchanges in rpt-1 expression as described supra. For example, total RNAin peripheral blood lymphocytes from a patient can be assayed for thepresence of Rpt-1 mRNA, where the presence or amount of Rpt-1 mRNA isindicative of a state of T cell activation associated with an immuneabnormality.

5.8.2. Therapy

The rpt-1 proteins, analogues, derivatives, and subsequences thereof,anti-rpt-1 antibodies, and Rpt-1 nucleic acids and subsequences thereofof the invention can be used for therapy of diseases or disordersassociated with expression of products encoded by sequences whosetranscription is controlled by an IL-2r promoter or a promoter of an LTRof a human lymphotropic retrovirus. For example, an Rpt-1 codingsequence can be incorporated into an hematopoietic stem cell for use ingene therapy of AIDS patients, where it will be expressed, causingdecreased expression of HIV-1-LTR-directed genes. In another embodiment,rpt-1 may be used to regulate gene expression of HTLV-I or HTLV-II inleukemic patients. The rpt-1 protein itself can be conjugated with ananti-nuclear protein antibody or anti-DNA antibody in order to effectproper targeting of the rpt-1 molecule.

Various delivery systems are known and can be used for therapeuticdelivery of rpt-1 and related molecules, e.g., encapsulation inliposomes, expression by bacteria, etc. Other methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, and intranasal routes.

In another embodiment of the invention, rpt-1, a related molecule, orantibody to rpt-1, which is shown to inhibit rpt-1 function, can be usedin an attempt to increase T cell activation, e.g., of a patient who isimmunosuppressed due to chemotherapy or radiation exposure.

6. CLONING AND CHARACTERIZATION OF THE RPT-1 GENE AND ITS ENCODEDPROTEIN

In the examples section herein, we describe a regulatory proteinT-lymphocyte-1 (Rpt-1) gene, selectively expressed by resting relativeto activated CD4⁺ helper/inducer T cells, which encodes a novelintracellular protein (41,000 molecular weight) that down-regulates geneexpression directed by the promoter region of the interleukin-2 receptoralpha chain (IL-2rα) gene and by the promoter region of the longterminal repeat of the human immunodeficiency virus type 1 (HIV-1). Ourdata suggest that rpt-1 levels may be inversely correlated withactivation of CD4⁺ T cells and HIV-1 replication leading to clinicalsymptoms of the acquired immune deficiency syndrome (AIDS).

In additional experiments, we have demonstrated specific regulation byrpt-1 of gene expression controlled by a promoter of HTLV-II or HTLV-I,but not by a promoter of herpes simplex virus thymidine kinase or SV40.

6 1 Materials and Methods 6.1.1. Cells

The derivation and maintenance of T cell clones used herein have beendescribed previously (Clayberger, C., et al., 1983, J. Exp. Med.157:1906-1917; Clayberger, C., et al., 1984, J. Exp. Med.158:1881-1894). Briefly, Cl.Ly1-T1 and Cl.Ly1-N5 are inducer T cellclones (id.). C1.NK-11 is a natural killer cell clone (Nabel, G., etal., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1157-1161) and Cl.Ly23.4 isa suppressor T cell clone (Fresno, M., et al., 1981, J. Exp. Med.153:1260-1274). Ar-5 is an arsonate-reactive inducer T cell clone (Rao,A., et al., 1983, J. Exp. Med. 159:1243-1258). Ar-5v is a variant ofAr-5 that constitutively expresses high levels of IL-2rα (as measured byimmunofluorescence using a monoclonal antibody, AMT-13, to the IL-2rαchain, supplied by Boehringer Mannheim). Ar-5v, but not Ar-5, isactivated by recombinant IL-2 (Genzyme) in the absence of antigen;activation is completely blocked by AMT-13. Ar-5v does not producedetectable levels of IL-2 unless activated by IL-2 or antigen and IA^(d)macrophages. Jurkat (Nabel, G. and Baltimore, D., 1987, Nature326:711-713) and EL-4 are human and murine T cell lines, respectively,and COS7m6 is an SV40-transformed monkey kidney epithelial cell line(Okayama, H., and Berg, P., 1983, Mol. Cell. Biol. 3:280-289).

6.1.2. Activation of T Cells

Resting T cell clones were activated by antigen (Clayberger, C., et al.,1983, J. Exp. Med. 157:1906-1917; Clayberger, C., et al., 1984, J. Exp.Med. 158:1881-1894; Nabel, G., et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1157-1161; Fresno, M., et al., 1981, J. Exp. Med.153:1260-1274) or by addition of 5 ug/ml of concanavalin A (ConA) and 10ng/ml of 12-0-tetradecanoylphorbol 13-acetate (TPA). Jurkat cells wereactivated by the addition of TPA (40 ng/ml) and phytohemagglutinin (PHA)(10 ug/ml). EL-4 cells were activated by the same amounts of TPA and PHAin addition to calcium ionophore A23187 (5 ug/ml).

6.1.3. Production of a T Cell Probe

Poly(A)⁺ RNA from L cells (a fibroblast tumor) and from 2PK3 (a B celllymphoma) was prepared and hybridized to ³² P-labelled cDNA obtainedfrom Cl.Ly1-TI cells (22 hours after activation) as described previously(Chirgwin, J., et al., 1979, Biochemistry 18:5294-5299; Freeman, G. J.,et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4094-4098). The remainingsingle-stranded cDNA purified by hydroxyapatite chromatography washybridized with poly(A)⁺ RNA from MOPC 315 (a B cell myeloma) and thesingle-stranded fraction was again isolated by hydroxyapatitechromatography.

6.1.4. Construction of a T Cell cDNA Library

Poly(A)⁺ RNA from Cl.Ly1-T1 (22 hours after activation) was used toprepare a cDNA library of 3.8×10⁵ independent clones in the pcD vector(Okayama, H. and Berg, P., 1982, Mol. Cell. Biol. 2:161-170; Okayama, H.and Berg, P., 1983, Mol. Cell. Biol. 3:280-289). 11,300 colonies, from a0.5 to 20 kb cDNA insert size-selected sublibrary were sparsely platedon nitrocellulose and then probed (Maniatis, T., et al., 1982, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.) with the T cell specific probe described above. Positivelyhybridizing colonies were picked for further analysis.

6 1.5. Nucleic Acid Blotting and Hybridization

RNA slot and Northern blots were performed as described (Chirgwin, J.,et al., 1979, Biochemistry 18:5294-5299). The same amount of total RNAwas used for every sample of a given experiment. Southern blots werecarried out as described by Gatt et al. (1984, Biotechniques 2:148-155).

6.1.6. Western Blot Analysis

Mouse spleen cells (10⁷ -10⁸) were washed twice with PBS and resuspendedin 1 ml of lysis buffer (1% NP-40, 0.02% NaN₃ in PBS), 10 ul ofphenylmethylsulfonyl fluoride (PMSF) (34.8 mg in 1 ml methanol) and 20ul iodoacetamide (36.2 mg in 0.5 ml distilled H₂ O). The mixture wasincubated on ice for 30 minutes and loaded onto a polyacrylamide gel.After transfer of proteins from the polyacrylamide gels tonitrocellulose sheets (Towbin, H., et al., 1979, Proc. Natl. Acad. Sci.U.S.A. 76:4350-4354), the filters were incubated for one hour withpro-binding protein (5% milk protein, 1% fetal calf serum in Tris salineazide (TSA)) and rabbit IgG (1:200 serum dilution) obtained afterimmunization with keyhole limpet hemocyanin (KLH) conjugated to asynthetic peptide corresponding to the most hydrophilic portion of therpt-1 protein (KKEKKE) (FIG. 3), followed by a 30 minute incubation withan ¹²⁵ I-labelled goat anti-rabbit antibody. Filters were washed withTSA and exposed for autoradiography.

6.1.7. Plasmids

230 of 11,300 colonies from the size selected cDNA library (Shen, F.-W.,et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:7360-7363) describedsupra hybridized with the T cell cDNA probe described above. PlasmidpcD-rpt-1 was obtained from a hybridizing colony. pcD-rpt-1 DNAhybridized to a 3.7 kb RNA expressed in T cells (see Section 6.2).pcD-rpt1fs is a frame-shift mutant of pcD-rpt1 obtained by digestingpcD-rpt1 with the enzyme BstEII, blunt-ending with T4 polynucleotidekinase, and religating. The mutation was confirmed by DNA sequencing.pSV2CAT contains the SV40 enhancer-promoter region upstream of thebacterial chloramphenicol acetyl transferase (CAT) gene (Gorman, C. M.,et al., 1982, Mol. Cell. Biol. 2:1044-1051). The plasmid IL2rpCATcontains the human IL-2rα promoter region upstream of CAT (Leonard, W.J., et al., 1985, Science 230:633-639). The plasmid pLTR-1CAT containsthe U3 and R regions of the HIV-1 long terminal repeat (LTR)(nucleotides -463 to + 80) (Tong-Starksen, S. E., et al., 1987, Proc.Natl. Acad. Sci. U.S.A. 84:6845; Sanchez-Pescador, R., et al., 1985,Science 227:484-492). pSV7fdtat is an expression vector for the tatprotein of HIV-1 (id.).

6.1.8. Transfection of Cell Lines and Chloramphenicol Acetyl TransferaseAssays

The DEAE-dextran technique (Queen, C. and Baltimore, D., 1983, Cell33:741-748) was used to transfect adherent cells (10⁶cells/transfection) and cells grown in suspension (10⁷cells/transfection). 48 hours after transfection, CAT lysates wereprepared by freeze-thawing (Gorman, C. M., et al., 1982, Mol. Cell.Biol. 2:1044-1051). Assays were done with equivalent amounts of proteinfrom each lysate (approximately 200-300 ug protein per assay) asdescribed (id.), except that the final acetyl coenzyme A concentrationin the reaction mix was 3 mM. Reaction mixes were evaluated for percentconversion of chloramphenicol to acetylated forms, at different timepoints, by thin layer chromatography. Experiments were done in thelinear range of the assay, and were repeated three to five times withdifferent batches of purified DNA. Extracts from cells that had not beentransfected with CAT plasmids showed no CAT activity. Expression of theRpt-1 cDNA insert was confirmed at both the protein level (by FACS) andthe RNA level (by slot blots using the SV40 polyadenylation signalregion from the pcD vector as hybridization probe, to avoidhybridization with endogenous rpt-1 in T cells). COS and Jurkat cellstransfected with pcD-rpt1 expressed the recombinant protein while thosetransfected with pcD-rpt1fs did not. Activation of Jurkat cells did notsignificantly change the levels of the recombinant protein according toFACS analysis. Southern blot analysis revealed that equivalent amountsof plasmid were transfected in every case.

6.1.9. Immunofluorescence

rpt-1: Cells were placed on coverslips, incubated with 0.5%glutaraldehyde for 30 seconds, washed, and incubated with the IgGfraction of a rabbit anti-KKEKKE antiserum at 1:200 final concentration,at 4° C. for 40 minutes. The cells were then washed twice with 1 X PBSbefore addition of rhodamine-conjugated goat anti-rabbit IgG (1:100dilution) (Cooper Biomedical, Glendale, Calif.).

Cell surface antigens: Thy1⁺ and Ig⁺ spleen cells (Fresno, M., et al.,1982, Cell 30:707-713; Clayberger, C., et al., 1984, J. Exp. Med.158:1881-1894; Fresno, M., et al., 1981, J. Exp. Med. 153:1260-1274)were incubated with either AMT-13, L3T4, or Ly2 monoclonal antibodies(Becton-Dickinson, Mountainview, Calif.), washed thrice and incubatedwith fluorescein isothiocyanate (FITC)-goat anti-rat IgG (1:200dilution) (Cappel, Westchester, Pa.). The intensity of fluorescence ofeach cell population analyzed by FACS is presented on a log₁₀ scale(FIGS. 4, 6). Where indicated, cells were visualized using a fluorescentphotomicroscope (Olympus IMT2) with barrier filters of 420 to 490 nm forfluorescein, and 500 to 590 nm for rhodamine.

6.2. Results

After activation by antigen or by the T cell mitogen concanavalin A, theT cell clone Cl.Ly1-T1 undergoes one or two rounds of division andsecretes inducer-specific proteins (Nabel, G., et al., 1981, Cell23:19-28; Clayberger, C., et al., 1984, J. Exp. Med. 158:1881-1894). 230of the 11,300 colonies from a size-selected Cl.Ly1-T1 pcD cDNA libraryhybridized to a cDNA probe enriched for genes expressed in T cells. Oneof these inserts, termed Rpt-1 (regulatory protein T cell), hybridizedto a 3.7 kb mRNA present in inducer T cell clones (FIG. 1A, lanes e-h),but not in a suppressor T cell clone (FIG. 1A, lanes c,d), a naturalkiller cell clone (FIG. 1A, lane b), or a cytolytic T cell clone (FIG.1A, lane a). Expression was not dependent on long-term growth of T cellclones since the Rpt-1 probe hybridized to a 3.7 kb RNA from freshlyexplanted lymphoid cells of the spleen and thymus (FIG. 1B).

Resting inducer T cells (FIG. 1A, lanes e,g) showed higher levels ofRpt-1 RNA than activated inducer T cells (FIG. 1A, lanes f,h). Analysisof the time course of Rpt-1 expression during activation of Cl.Ly1-T1with antigen and splenic adherent cells showed that Rpt-1 transcriptionwas not detectable four to eight hours after activation (upper panel,FIG. 1C). Expression of other genes we have studied, including Tcell-specific genes, is either unchanged or, more commonly, increasedafter activation, as illustrated by Northern blot analysis ofgamma-interferon RNA (lower panel, FIG. 1C).

The complete nucleotide sequence of the full length Rpt-1 clone cDNAinsert was determined using the Maxam and Gilbert method (Maxam, A. M.,and Gilbert, W., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:560-564). Thetotal sequence of 3700 bp contains an open reading frame 353 amino acidslong (positions 165 to 1226) (FIG. 2), followed by a very long3'-untranslated region of 2466 bp with several potential polyadenylationsignals. The first methionine in this open reading frame is encoded by asubsequence that fits the consensus for eukaryotic translationinitiation signals (Kozak, M., 1984, Nucl. Acids Res. 12:857-872). Thepredicted protein has a molecular weight of 41,330 daltons, isrelatively neutral in charge (pI of 6.29), and includes a potentialN-linked glycosylation site (amino acid residue numbers 30 to 32).However, the lack of an obvious signal sequence or membrane spanningregion (Sabatini, D. D., et al., 1982, J. Cell. Biol. 92:1-19) makes itunlikely to be a membrane-bound protein.

Additional experiments have indicated the existence of a "silencer"sequence in the 3' untranslated region of the Rpt-1 gene, to which a Tcell-specific protein binds, thereby decreasing transcription levels ofRpt-1 mRNA. The strain-specific polymorphism associated with differentlevels of Rpt-1 expression (FIG. 5) resides within the silencer region,as judged by Southern blot hybridization using a cDNA probecorresponding to a portion of the 3' untranslated region of the Rpt-1gene.

Western blot analysis (as described in Section 6.1.6) of mouse spleencell extracts using radiolabeled antibodies raised to a syntheticpeptide (KKEKKE) that corresponds to the predicted most hydrophilicregion of the rpt-1 protein (Hopp, T., and Woods, K., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:3824-3828) (FIGS. 2,3), revealed a protein of theexpected molecular weight (FIG. 4A). Immunofluorescent analysis withfluorescein-conjugated anti-KKEKKE antibody was used to determine theexpression of rpt-1 in different lymphoid lineages (see Section 6.1.9).Less than 0.3% of splenic Ig⁺ cells (B cells), less than 2% of Ly2⁺ Tcells, and 78-91% of L3T4⁺ cells were positive for rpt-1.

The carboxyl-terminal half of rpt-1 has a subsequence (TVPQKRKRT; aminoacid residue numbers 268 to 276, FIG. 2) similar to the nuclearlocalization signal of the simian virus 40 (SV40) large T antigen (FIG.3) (Kalderon, D., et al., 1984, Cell 39:499-509), which may interactwith a receptor in the nuclear envelope (Goldfarb, D. S., et al., 1986,Nature 322:641-644). COS7m6 cells transfected with the expression vectorpcD-rpt1 , but not with pcD-rpt1fs (a frame shift mutant of rpt-1, seeSection 6.1.7), were positive for rpt-1 expression, as measured byimmunofluorescence using anti-KKEKKE antibody and detected by FACSanalysis (FIG. 4B); microscopic observation showed predominantly nuclearstaining (FIG. 4C).

The amino-terminal half of the rpt-1 protein contains pairs of cysteineresidues (FIGS. 2 and 3) in the following pattern: Cys-X₂ -Cys-X₁₆-Cys-X₂ -Cys-X₁₆ -Cys-X₂ Cys. These cysteine residues are unlikely to beinvolved in disulfide bridge formation in view of conditions in theintracellular environment. This organization of cysteine residues is aprominent feature of proteins involved in metal binding and regulationof gene expression (reviewed in Berg, J., 1986, Science 232:485-487).The cysteine residues are believed to bind metals such as zinc while theintervening amino acids protrude in a finger-like projection. The rpt-1protein has the potential to form two alternative fingers (FIG. 3).Secondary structure algorithms (Hopp, T. and Woods, K., 1981, Proc.Natl. Acad. Sci. U.S.A. 78:3824-3828; Chou, P. Y. and Fasman, G. D.,1978, Ann. Rev. Biochem. 47:251-276) predict that the regions containingthe cysteine pairs are hydrophobic and in a beta sheet configuration,while the intervening segments representing finger-like projections arehydrophilic and include a beta-turn (FIG. 3). There is an additionalpair of cysteine residues in the rpt-1 protein followed by a pair ofhistidines with the potential to form a metal finger (FIG. 3).

To determine whether the Rpt-1 gene maps to any of the loci that arethought to affect T cell function, we located the genetic linkage ofRpt-1 using restriction fragment length polymorphisms (RFLP) present inrecombinant inbred strains derived from C57B1/6 (B) and DBA (D) strains.Genomic DNA was digested with HindIII, and the 5' 1.3 kb Rpt-1 codingregion insert (RsaI-XbaI fragment) was used as a hybridization probe.Since the B strain DNA digest shows a major band of 10 kb and minorbands of 9.5, 4, 2, 1, and 0.8 kb, whereas the D strain DNA shows amajor band of 9 kb and minor bands of 12, 10, and 1 kb, each BXD straincould be unambiguously assigned a parental origin and correlated withthe genetic maps of the 26 BXD strains. This analysis indicated linkageto HBB, the hemoglobin b chain locus on chromosome 7 (FIG. 5).

A polymorphism that affects expression of IL-2rα on L3T4⁺ inducer Tcells (but not Ly2⁺ T cells) also maps to the HBB locus using BXD lines(Kawamura, H., et al., 1986, J. Exp. Med. 163:1376-1390). Analysis ofthe relationship between expression of rpt-1 and IL-2rα on different Tcell clones showed an inverse correlation (FIG. 6A). Moreover, T cellstransfected with pcD-rpt1, but not pcD-rpt1fs cDNA, pcD, orpcD-beta-galactosidase, showed reduced levels of IL-2rα (assessed byFACS analysis, FIG. 6A); slot blot analysis of these transfectantsconfirmed a decrease in IL-2r mRNA while levels of IL-3 mRNA wereunchanged. The experiments described infra were performed to determinewhether inhibition of IL-2rα expression after Rpt-1 transfectionreflected a direct effect of rpt-1 on IL-2rα gene expression.

Co-transfection of pcD-rpt1, compared with pcD-rpt1fs (FIG. 6B, lanes1,2; see Section 6.1.8), pcD, or pcD-betagalactosidase, resulted in a3-5 fold down-regulation of gene expression directed by the human IL-2rαpromoter in COS7m6 cells (FIG. 6B, lanes 1,2). In contrast, pcD-rpt1transfection had no effect on gene expression directed by the SV40enhancer-promoter (plasmid pSV₂ CAT) (FIG. 6B, lanes 5,6) and theadenoviral thymidine kinase promoter.

We investigated the effects of rpt-1 on other inducer-specific cellularand retroviral genes. Co-transfection of pcD-rpt1 , but not pcD-rpt1fs,resulted in a three-fold inhibition of HIV-1 LTR-directed geneexpression in COS7m6 cells (FIG. 6B, lanes 3,4). We also performed thesecotransfection assays with T lymphocytes. Resting T cells containendogenous rpt-1, and the HIV-1 LTR is expressed at near backgroundlevels in these cells (Nabel, G. and Baltimore, D., 1987, Nature326:711-713; Tong-Starksen, S. E., et al., 1987, Proc. Natl. Acad. Sci.U.S.A. 84:6845); the low baseline level of HIV-1 LTR expression makes itdifficult to detect inhibition of expression after transfection ofexogenous cDNA. Therefore, we tested the effect of rpt-1 on HIV-1LTR-directed gene expression in the presence of the HIV-1trans-activator protein, tat. A three-fold inhibition was observed inresting Jurkat and EL-4 cell lines in the presence of HIV-1 tat (FIG.6B). However, no inhibition of HIV-1 LTR-directed gene expression, inthe presence or absence of HIV-1 tat, was seen after activation of theseT cell lines (FIG. 6B).

In cotransfection experiments similar to those described supra, we havedemonstrated specific regulation by rpt-1 of gene expression controlledby the promoters of the human retroviruses HTLV-I and HTLV-II, but notby the promoters of herpes simplex virus thymidine kinase or of SV40.

6.3. Discussion

Potential metal-binding fingers in the amino-terminal half (reviewed inBerg, J., 1986, Science 232:485-487) and a stretch of predominantlycharged amino acids in the carboxy-terminal portion (Legrain, M., etal., 1986, Nucl. Acids Res. 14:3059-3073; Keegan, L., et al., 1986,Science 231:699-704; Hope, J. and Struhl, K., 1986, Cell 46:885-894) arefeatures of several proteins that regulate gene expression. The markeddifference of character between the amino- and carboxy-terminal halvesof the rpt-1 protein may denote at least two functional domains, as inthe case of Xenopus transcription factor IIIA (Miller, J., et al., 1985,EMBO J. 4:1609-1614): an amino-terminal domain that targets the proteinto a particular set of nucleic acids and a carboxy-terminal domain thatexerts regulatory activity. Rpt-1 may affect IL-2rα and HIV-1 expressionthrough cis-acting negative regulatory elements or through competitionwith proteins that bind to enhancer or activator sequences. rpt-1 maydirectly bind to regions of the IL2rα and HIV-1 promoter, such as thosesequences that are reported to be similar (Fugita, T., et al., 1986,Cell 46:401-407).

We have determined, by S1 mapping analysis, that decreased levels ofgene expression directed by the human IL-2rα promoter can be ascribed toa decrease in full-length RNA levels (rather than a shift in thetranscriptional start site). Although decreased gene expression was onlythree to five fold as assessed by CAT assays, this inhibition wasphysiologically significant since a substantial fraction (approximately1/3) of cells transfected with rpt-1 no longer displayed surface levelsof IL-2rα required for activation by IL-2 (FIG. 6A; Lethe Bich-Thuy, etal., 1987, J. Immunol. 139:1550-1556). The regulation of human IL2-rα-and HIV-1, HTLV-I, and HTLV-II LTR-directed gene expression by rpt-1indicates that this recombinant product is biologically active acrossspecies. By FACS analysis of Jurkat cells (a human T cell line) with theanti-KKEKKE antibody, we have detected a related protein whose levelsare undetectable 16 hours after activation.

The hallmark of HIV-1 associated diseases is a relatively longasymptomatic period concurrent with evidence for low level persistentinfection (Klatzmann, D. and Gluckman, J. C., 1986, Immunol. Today7:291-296). One explanation of HIV-1 latency comes from low levels ofNF-Kappa B (Nuclear Factor Kappa B; Nabel, G. and Baltimore, D., 1987,Nature 326:711-713; Tong-Starksen, S. E., et al., 1987, Proc. Natl.Acad. Sci. U.S.A. 84:6845) in resting T cells. Activated T cells containincreased levels of the NF-Kappa B factor or its functional equivalent,which interacts with the HIV-1 enhancer sequence to induce elevatedlevels of HIV-1 LTR-directed gene expression (id.). The observed failureof rpt-1 to down-regulate HIV-1 LTR-directed gene expression instimulated T cells suggests that NF-Kappa B is dominant in the activatedstate.

The expression of the Rpt-1 gene in resting CD4⁺ T cells provides anadditional basis for the latent state of HIV-1. rpt-1 directly inhibitsHIV-1 gene expression even in the presence of significant amounts of tatprotein (FIG. 6B), and prevents expression of surface levels of IL-2rαrequired for efficient IL-2 mediated activation (Lethe Bich-Thuy, etal., 1987, J. Immunol. 139:1550-1556) (FIG. 6, A and B). A consequenceof these activities should be limitation of HIV-1 mediated destructionto clones of CD4⁺ cells that are specifically activated by antigenicdeterminants, after exposure of the immune system to bacteria andviruses. Expression of Rpt-1 in the remaining CD4⁺ clones may preemptsecondary activation by IL-2 and efficient expression of HIV-1, whichmay account for the slow decay of CD4⁺ T cells despite chronic HIV-1infection. It is possible that rpt-1 levels in inducer T cells may beinversely correlated with increased HIV-1 replication responsible forthe clinical symptoms of AIDS.

7. DEPOSIT OF MICROORGANISMS

E. coli strain MC1061 carrying plasmid pcD-rpt-1 has been deposited withthe Agricultural Research Culture Collection (NRRL), Peoria, Ill., andhas been assigned accession number B-18297.

The present invention is not to be limited in scope by themicroorganisms deposited since the deposited embodiment is intended as asingle illustration of one aspect of the invention and anymicroorganisms which are functionally equivalent are within the scope ofthis invention. Indeed, various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

It is also to be understood that all base pair sizes given fornucleotides are approximate and are used for the purpose of description.

What is claimed is:
 1. An isolated and purified DNA sequence as shown inFIG.
 2. 2. The sequence of claim 1 which comprises a cDNA sequence. 3.The sequence of claim 1 which comprises a genomic DNA sequence.
 4. AnRNA sequence complementary to the DNA sequence of FIG.
 2. 5. An isolatedand purified DNA sequence comprising a sequence substantially homologousto the nucleotide sequence shown in FIG. 2 or a portion thereof whichencodes a protein or polypeptide which is capable of decreasing theexpression of genes under the control of the interleukin 2 receptoralpha chain promoter or the long terminal repeat of the humanimmunodeficiency virus type
 1. 6. A recombinant vector containing theDNA sequence of claim
 1. 7. A recombinant vector containing the DNAsequence of claim
 5. 8. The recombinant DNA vector comprising pcD-rpt-1.9. An isolated cell containing the vector of claim 6 or
 7. 10. Anisolated cell containing the vector of claim
 8. 11. The isolated cell ofclaim 9 which is mammalian.
 12. The isolated cell of claim 10 which ismammalian.
 13. The isolated cell of claim 9 which comprises a bacterium.14. The isolated cell of claim 10 which comprises a bacterium.
 15. Thebacterium of claim 14 comprising an Escherichia coli as deposited withthe NRRL and assigned accession number B-18297, or a mutant, orgenetically engineered derivative thereof which encodes a protein orpolypeptide which is capable of decreasing the expression of genes underthe control of the interleukin 2 receptor alpha chain promoter or thelong terminal repeat of the human immunodeficiency virus type
 1. 16. Amethod for regulating gene expression controlled by a promoter of theinterleukin-2 receptor alpha chain gene comprising (1) introducing anucleic acid sequence encoding the rpt-1 protein as shown in FIG. 2 orbiologically active fragment thereof into a host cell, and (2)subjecting said host cell to conditions such that the rpt-1 protein isexpressed in amounts sufficient to regulate said gene expression withinsaid host cell.
 17. A method for regulating gene expression controlledby a promoter of a long terminal repeat of a human lymphotropicretrovirus comprising (1) introducing a nucleic acid sequence encodingthe rpt-1 protein as shown in FIG. 2 or a biologically active fragmentthereof into a cell infected by a human lymphotropic retrovirus, and (2)subjecting said host cell to conditions such that the rpt-1 protein isexpressed in amounts sufficient to regulate said gene expression withinsaid host cell.
 18. The method according to claim 17 in which theretrovirus comprises human immunodeficiency virus type I.
 19. The methodaccording to claim 17 in which the retrovirus comprises human T cellleukemia virus type I.
 20. The method according to claim 17 in which theretrovirus comprises human T cell leukemia virus type II.
 21. The methodaccording to claim 16 in which the regulation results in a decrease inthe level of gene expression.
 22. The method according to claim 17 inwhich the regulation results in a decrease in the level of geneexpression.
 23. The method according to claim 18 in which the regulationresults in a decrease in the level of gene expression.